Participants and procedures
The MOM-COPE is a longitudinal and multi-centric cohort study that involves ten neonatal units in Northern Italy. The fully detailed description of this project is reported elsewhere.25 Here we report on a sample of 108 (62%) mother-infant dyads who provided complete data for prenatal (T0), neonatal (T1) and 3-month (T2) assessments by February 2021 (Figure 1). Mothers were included if at least 18-year-old, in the absence of prenatal and perinatal diseases or injuries, if they delivered at term (i.e., from 37+0 to 41+6 weeks of gestation), and if they were negative for COVID-19 at delivery. Mothers were first contacted at antepartum classes or immediately following the postpartum period. Socio-demographic and neonatal data were obtained from medical records. Within 48 hours from delivery, the mothers filled in a first set of questionnaires to provide retrospective quantitative measures of prenatal COVID-19-related stress. Between 6 and 24 hours, buccal cells were obtained from mothers and infants using DNA Genotek Oragene OC-175, according to manufacturer guidelines. When infants were approaching 3-month-age, mothers filled-in a questionnaire on infants’ temperament. The study was approved by the Ethics Committees of the project lead institution (IRCCS Mondino Foundation, Pavia, Italy) and the participating hospitals. All the procedures were performed in accordance with the 2018 Declaration of Helsinki. All mothers provided informed consent to participate to the study.
Prenatal measures, T0
Mothers self-reported on socio-demographic (i.e., age, educational level, occupational status). At delivery mothers retrospectively reported on their prenatal COVID-19-related stress during the last trimester of pregnancy through an ad-hoc questionnaire (Supplementary File S1). A mean COVID-19-related prenatal stress score was obtained, ranging from 1 (low) to 5 (high).
Neonatal measures, T1
Neonatal characteristics (i.e., gestational age, birth weight, head circumference, neonatal length, Apgar at minute 1, breastfeeding at birth, and mode of delivery) were collected from medical records. The saliva was collected from both mothers and infants using the OraCollect for Pediatrics kit OC-175 (DNA Genotek, Ottawa, Canada) between six and twenty-four hours from delivery. Methylation assessment was conducted according to previous validated procedures from this lab.36,37 The genomic DNA was extracted following manufacturer’s protocols and its quality was assessed using a Qubit fluorometer Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA). The methylation status of the SLC6A4 gene’s region (chr17:28562750–28562958, 13 CpGs) was assessed by PCR amplification of bisulfite-treated DNA followed by Next Generation Sequencing (NGS) on a NEXTSeq-500 (Illumina, San Diego, California, USA).
Postnatal measures, T2
At infants’ 3-month-age, infants’ temperament was assessed with the short form version of the Infant Behaviour Questionnaire-Revised, IBQ-R.38 The IBQ-R includes 91 items that are rated on a 7-point and summarized into 14 scales and 3 main factors: surgency (i.e., activity level and expression of pleasure), negative affectivity (i.e., distress, sadness and fear) and regulatory capacity (i.e., cuddliness, soothability and orienting).
Plan of analysis
Descriptive statistics were computed for socio-demographic characteristics and for all the variables reported in the Measures section. Parametric indexes (i.e, mean, standard deviation, range) were obtained for continuous variables, whereas frequencies and percentages were obtained for categorical variables. Normal distribution was checked for all variables of interest and normalization occurred by means of natural log (ln) adjustment. Bivariate Pearson’s correlation coefficients were computed to assess the association among COVID-19-related prenatal stress and both infants and mothers’ methylation status of SLC6A4 CpG sites. Multiple-comparison bias was checked using the Benjamini-Hochberg procedure, q < .10.39,40 A principal component analysis (PCA) was used to reduce the number of methylation items; a single principal component (PC1) accounted for 35% of the total variance in SLC6A4 methylation and it was used in further analyses. Bivariate correlations were carried to test for the potential association of neonatal and socio-demographic variables with both SLC6A4 PC1 methylation and infants’ surgency at 3 months. A linear general model was used to estimate the association between COVID-19-related prenatal stress and infants SLC6A4 PC1 methylation (ln). A linear general model was used to estimate the association between infants SLC6A4 PC1 methylation and 3-month surgency score. Analyses were carried using R and IBM SPSS 26 for Windows, with p< .05.